Native incretins prevent the development of atherosclerotic lesions in apolipoprotein E knockout mice.

AIMS/HYPOTHESIS: Several lines of evidence suggest that incretin-based therapies suppress the development of cardiovascular disease in type 2 diabetes. We investigated the possibility that glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) can prevent the development of atherosclerosis in Apoe (-/-) mice. METHODS: Apoe (-/-) mice (17 weeks old) were administered GLP-1(7-36)amide, GLP-1(9-36)amide, GIP(1-42) or GIP(3-42) for 4 weeks. Aortic atherosclerosis, oxidised LDL-induced foam cell formation and related gene expression in exudate peritoneal macrophages were determined. RESULTS: Administration of GLP-1(7-36)amide or GIP(1-42) significantly suppressed atherosclerotic lesions and macrophage infiltration in the aortic wall, compared with vehicle controls. These effects were cancelled by co-infusion with specific antagonists for GLP-1 and GIP receptors, namely exendin(9-39) or Pro(3)(GIP). The anti-atherosclerotic effects of GLP-1(7-36)amide and GIP(1-42) were associated with significant decreases in foam cell formation and downregulation of CD36 and acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in macrophages. GLP-1 and GIP receptors were both detected in Apoe (-/-) mouse macrophages. Ex vivo incubation of macrophages with GLP-1(7-36)amide or GIP(1-42) for 48 h significantly suppressed foam cell formation. This effect was wholly abolished in macrophages pretreated with exendin(9-39) or (Pro(3))GIP, or with an adenylate cyclase inhibitor, MDL12,330A, and was mimicked by incubation with an adenylate cyclase activator, forskolin. The inactive forms, GLP-1(9-36)amide and GIP(3-42), had no effects on atherosclerosis and macrophage foam cell formation. CONCLUSIONS/INTERPRETATION: Our study is the first to demonstrate that active forms of GLP-1 and GIP exert anti-atherogenic effects by suppressing macrophage foam cell formation via their own receptors, followed by cAMP activation. Molecular mechanisms underlying these effects are associated with the downregulation of CD36 and ACAT-1 by incretins.

Coping with the inevitable: how cells repair a torn surface membrane.

Disruption of the cell plasma membrane is a commonplace occurrence in many mechanically challenging, biological environments. 'Resealing' is the emergency response required for cell survival. Resealing is triggered by Ca2+ entering through the disruption; this causes vesicles present in cytoplasm underlying the disruption site to fuse rapidly with one another (homotypically) and also with the adjacent plasma membrane (heterotypically/exocytotically). The large vesicular products of homotypic fusion are added as a reparative 'patch' across the disruption, when its resealing requires membrane replacement. The simultaneous activation of the local cytoskeleton supports these membrane fusion events. Resealing is clearly a complex and dynamic cell adaptation, and, as we emphasize here, may be an evolutionarily primitive one that arose shortly after the ancestral eukaryote lost its protective cell wall.

Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization.

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.

Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum.

The calcium indicator dye fluo-3/AM was loaded into the ER of isolated cortices of unfertilized eggs of the sea urchin Arbacia punctulata. Development of the fluorescent signal took from 8 to 40 min and usually required 1 mM ATP. The signal decreased to a minimum level within 30 s after perfusion with 1 microM InsP3 and increased within 5 min when InsP3 was replaced with 1 mM ATP. Also, the fluorescence signal was lowered rapidly by perfusion with 10 microM A23187 or 10 microM ionomycin. These findings demonstrate that the cortical ER is a site of ATP-dependent calcium sequestration and InsP3-induced calcium release. A light-induced wave of calcium release, traveling between 0.7 and 2.8 microns/s (average speed 1.4 microns/s, N = 8), was sometimes observed during time lapse recordings; it may therefore be possible to use the isolated cortex preparation to investigate the postfertilization calcium wave.

Visualization of calcium transients controlling orientation of ciliary beat.

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.

Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection.

Previous studies suggest that kinesin heavy chain (KHC) is associated with ER-derived membranes that accumulate in the mitotic apparatus in cells of early sea urchin embryos (Wright, B. D., J. H. Henson, K. P. Wedaman, P. J. Willy, J. N. Morand, and J. M. Scholey. 1991. J. Cell Biol. 113:817-833). Here, we report that the microinjection of KHC-specific antibodies into these cells has no effect on mitosis or ER membrane organization, even though one such antibody, SUK4, blocks kinesin-driven motility in vitro and in mammalian cells. Microinjected SUK4 was localized to early mitotic figures, suggesting that it is able to access kinesin in spindles. In contrast to KHC-specific antibodies, two antibodies that react with kinesin-like proteins (KLPs), namely CHO1 and HD, disrupted mitosis and prevented subsequent cell division. CHO1 is thought to exert this effect by blocking the activity of a 110-kD KLP. The relevant target of HD, which was raised against the KHC motor domain, is unknown; HD may disrupt mitosis by interfering with an essential spindle KLP but not with KHC itself, as preabsorption of HD with KHC did not alter its ability to block mitosis. These data indicate that some KLPs have essential mitotic functions in early sea urchin embryos but KHC itself does not.

Microtubules and the endoplasmic reticulum are highly interdependent structures.

The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate filaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell. Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments. We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system.

Large plasma membrane disruptions are rapidly resealed by Ca2+-dependent vesicle-vesicle fusion events.

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390-393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an approximately 40 by 10 microm surface patch was torn off, entry of fluorescein stachyose (FS; 1, 000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (approximately 5-10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was approximately 3 mM Ca2+ (SW is approximately 10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+ -free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events.

Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs.

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.

Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg.

During the first cell cycle of the ascidian egg, two phases of ooplasmic segregation create distinct cytoplasmic domains that are crucial for later development. We recently defined a domain enriched in ER in the vegetal region of Phallusia mammillata eggs. To explore the possible physiological and developmental function of this ER domain, we here investigate its organization and fate by labeling the ER network in vivo with DiIC16(3), and observing its distribution before and after fertilization in the living egg. In unfertilized eggs, the ER-rich vegetal cortex is overlaid by the ER-poor but mitochondria-rich subcortical myoplasm. Fertilization results in striking rearrangements of the ER network. First, ER accumulates at the vegetal-contraction pole as a thick layer between the plasma membrane and the myoplasm. This accompanies the relocation of the myoplasm toward that region during the first phase of ooplasmic segregation. In other parts of the cytoplasm, ER becomes progressively redistributed into ER-rich and ER-poor microdomains. As the sperm aster grows, ER accumulates in its centrosomal area and along its astral rays. During the second phase of ooplasmic segregation, which takes place once meiosis is completed, the concentrated ER domain at the vegetal-contraction pole moves with the sperm aster and the bulk of the myoplasm toward the future posterior side of the embryo. These results show that after fertilization, ER first accumulates in the vegetal area from which repetitive calcium waves are known to originate (Speksnijder, J. E. 1992. Dev. Biol. 153:259-271). This ER domain subsequently colocalizes with the myoplasm to the presumptive primary muscle cell region.

Calcium release at fertilization in starfish eggs is mediated by phospholipase Cgamma.

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCgamma, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCbeta, which is activated by a G protein, we injected starfish eggs with a PLCgamma SH2 domain fusion protein that inhibits activation of PLCgamma. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCgammaSH2 protein is a specific inhibitor of PLCgamma in the egg, since it did not inhibit PLCbeta activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCgamma SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cgamma by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo.

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.

Golgi tubule traffic and the effects of brefeldin A visualized in living cells.

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein-Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5-10 min until rapidly emptying Golgi contents into the ER within 15-30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

Diffusional mobility of Golgi proteins in membranes of living cells.

The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged from 3 x 10(-9) square centimeters per second to 5 x 10(-9) square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes, which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.

Dynamics of the endoplasmic reticulum and golgi apparatus during early sea urchin development.

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2-3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is approximately 30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-microm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation.

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.

A new model for nuclear envelope breakdown.

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

Cancer incidence in Hiroshima and Nagasaki, Japan, 1958-1987.

The Hiroshima and Nagasaki tumour registries, which have been in operation since 1958, are among the few population-based cancer registries in Japan. This analysis evaluated cancer incidence in Hiroshima and Nagasaki between 1958 and 1987. The overall age-adjusted (World Population Standard) cancer incidence has increased from 217 to 301 per 100,000 among males, and from 176 to 197 per 100,000 among females during the first 30 years of cancer registration. The most recent rates are intermediate to rates in other countries. Despite a gradual decrease, gastric cancer remained the most common malignancy among males and females throughout the surveillance period, accounting for 24% of all cancers by the late 1980s. The rate of liver cancer has increased dramatically among males during the past 20 years, with a 2-fold increase in incidence in the past 10 years alone. The populations of Hiroshima and Nagasaki now have among the highest rates of liver cancer in the world. Breast cancer incidence in Hiroshima and Nagasaki, in contrast, is among the lowest in the world, although incidence rates have doubled since the 1960s. Other common malignancies include cancers of the lung, colon and rectum among males and cancers of the colon, cervix and lung among females.

Hepatic arterial infusion chemotherapy combined with hyperthermia for metastatic liver tumors of colorectal cancer.

The efficacy of combined hepatic arterial infusion chemotherapy and hyperthermia for unresectable hepatic tumors of colorectal cancer was retrospectively compared with hepatic arterial infusion chemotherapy alone. Nine cases were treated with combination therapy and eight cases were treated with hepatic arterial infusion alone. The response rate in the combined hepatic arterial infusion/hyperthermia group was higher (44% v 25%) and the survival period longer (2-year survival, 35% v 12%) than in the group treated with hepatic arterial infusion alone. The toxicity was not severe, with the exception of gastrointestinal symptoms, suggesting that this combination of modalities is useful in the treatment of metastatic liver tumors.

Fibroblast growth factor-2 and -9 regulate proliferation and production of matrix metalloproteinases in human gliomas.

Fibroblast growth factor (FGF) signaling has been recognized in human gliomas. We tested the effect of FGF-2 and FGF-9 on the proliferation and the expression of matrix metalloproteinases (MMPs) and their inhibitor (TIMP-1) in vitro. Both FGFs showed mitogenic activity on U251MG and NMC-G1 cells. MMP-1 expression and collagenolytic activity of NMC-G1 but not of U251MG, and TIMP-1 expression of both cells were stimulated by FGFs. MMP-2 expression, gelatinolytic activity, and chemoinvasion on the matrigel were not altered. FGFs may regulate proliferation and microenvironmental factors independently in each glioma type.